Hepatitis C virus (HCV) accounts for greater than 90% of transfusion-associated hepatitis in the U.S. and it is the predominant form of hepatitis in adults over 40 years of age. Almost all of the infections result in chronic hepatitis and nearly 20% of those infected develop liver cirrhosis.
Molecular cloning of the HCV genome has been accomplished by isolating the messenger RNA (mRNA) from the serum of infected chimpanzees and preparing cDNA using recombinant methodologies. [Grakoui A. et al., 1993, J. Virol. 67: 1385-1395]. It is now known that HCV contains a positive strand RNA genome comprising approximately 9400 nucleotides, organization of which is similar to that of flaviviruses and pestiviruses. The genome of HCV, a (+)-stranded RNA molecule of .about.9.4 kb, encodes a single large polyprotein of about 3000 amino acids which undergoes proteolysis to form mature viral proteins in infected cells.
Cell-free translation of the viral polyprotein and cell culture expression studies have established that the HCV polyprotein is processed by cellular and viral proteases to produce the putative structural and nonstructural (NS) proteins. At least ten mature viral proteins are produced from the polyprotein by specific proteolysis. The order and nomenclature of the cleavage products are as follows: NH.sub.2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH [Grakoui et al., 1993, J. Virol. 67:1385-95; Hijikata et al., 1991, PNAS 88:5547-51; Lin et al., 1994, J. Virol. 68:5063-73]. The three amino-terminal putative structural proteins, C (capsid), E1, and E2 (two envelope glycoproteins), are believed to be cleaved by a host signal peptidase of the endoplasmic reticulum (ER). The host enzyme is also responsible for generating the amino terminus of NS2. The proteolytic processing of the nonstructural proteins are carried out by the viral proteases: NS2-3and NS3, contained within the viral polyprotein. The NS2-3 protease catalyzes the cleavage between NS2 and NS3. It is a metalloprotease and requires both NS2 and the protease domain of NS3.
The NS3 protease catalyzes the rest of the cleavages in the nonstructural part of the polyprotein. The NS3 protein contains 631 amino acid residues and is comprised of two enzymatic activities: the protease domain contained within amino acid residues 1-181 and a helicase AIPase domain contained within the rest of the protein [Kim et al., 1995, Biochem Biophys Res. Comm., 215:160-166]. The gene encoding the HCV NS3 protein has been cloned as disclosed in U.S. Pat. No. 5,371,017. The NS3 protease is a member of the serine class of enzymes using a His, Asp, Ser catalytic triad [Love et al., 1996, Cell 87:331-42; Kim et al., 1996, Cell 87:343-55; Yan et al., 1998, Protein Science 7:83747]. Mutation of the Ser residue abolishes cleavage of NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B substrates. The cleavage between NS3 and NS4A is intramolecular, whereas the cleavages at the NS4A/4B, 4B/5A,5A/5B sites occur in trans.
Experiments using transient expression of various forms of HCV NS polyproteins in mammalian cells have established that the NS3 serine protease is necessary but not sufficient for efficient processing of all of these cleavages. Efficient proteolytic processing at NS4A/4B, NS4B/5A, and NS5A/5B sites within the non-structural domain of hepatitis C virus requires a heterodimeric complex of the NS3 serine protease and the NS4A protein. [Bartenschlager et al. 1995J. Virol. 67:3835-3844; Failla et al., 1994, J. Virol. 68:3753-3760]. Smaller domains of the NS4A protein have been shown to be sufficient for activation of NS3 protease [Butkiewicz et al., 1996, Virology, 225: 328-338; Lin et al., 1995, J. Virol 69:4377-8].
Because the HCV NS3 protease cleaves the non-structural HCV proteins necessary for HCV replication, the NS3 protease can be a target for the development of therapeutic agents against the HCV virus. Numerous medium to high throughput assays have been developed for the purpose of detecting inhibitors of HCV NS3 protease.
One example of such an assay that can be used to discover NS3 protease inhibitors is the scintillation proximity assay (SPA). SPA technology involves the use of beads coated with scintillant. Bound to the beads are acceptor molecules such as antibodies, receptors or enzyme substrates which interact with ligands or enzymes in a reversible manner. In a typical protease assay, the substrate peptide is biotinylated at one end and the other end is radiolabelled with low energy emitters such as .sup.125 I or .sup.3 H. The labeled substrate is then incubated with the enzyme. Avidin coated SPA beads are then added which bind to the biotin. When the substrate peptide is cleaved by the protease, the radioactive emitter is no longer in proximity to the scintillant bead and no light emission takes place. Inhibitors of the protease will leave the substrate intact and can be identified by the resulting light emission which takes place in their presence.
Another type of assay which can be used to screen for and characterize protease inhibitors are those that involve the use of substrates containing chromophores or fluorophores to detect cleavage. In a typical assay, a peptide substrate is attached to a chromophore or fluorophore and brought into contact with a protease known to cleave the substrate. The chromophore or fluorophore is released upon cleavage by the protease, and gives rise to an increase in absorbance or fluorescence detectable continuously or at assay's end-point by a commercial spectrophotometer or spectrofluorometer at certain wavelengths characteristic of the free chromophore or fluorophore. Alternatively, fluorescence can be detected from a substrate in which the effect of a fluorescent acceptor (resonance energy transfer) or quencher car. be released upon protease cleavage.
Another type of assay useful in detecting and characterizing protease inhibitors is the fluorescence polarization assay [see, e.g., Jolley et al., 1996, Journal of Biomolecular Screening 1(1):33-381]. In this type of assay, a substrate comprising both P and P' regions of the protease cleavage site is attached to a high molecular weight (MW) molecule binding moiety at one end, and a fluorophore molecule at the other end. Cleavage by the protease results in the separation of the side to which the high molecular weight binding site has been attached from the side to which the fluorophore molecule has been attached. Upon addition of a high molecular weight molecule which will bind to the high MW molecule binding moiety, the overall fluorescence polarization will decrease compared to that of no cleavage or an inhibited reaction. This method tolerates compounds that absorb light in the excitation and/or emission wavelength regions of the fluorophore.
A number of different chromogenic substrates are known. For example, para-nitroanilide (pNA) based substrates are widely known and used in various protease assays. HCV NS3 peptide substrates containing pNA have also been published [Landro et al., 1997, Biochemistry 36:9340-48; WO 97/19103], and made commercially available [Ac-EEVVAC-pNA from BACHEM]. Nitrophenyl esters have also been used to investigate the mechanism of action of serine proteases [e.g. Jersey et al., 1969, Biochemistry 8(5):1959-66; Williams, 1975, Journal of Chemical Society Perkin II, 947-53].
There are also a number of publications on the use of fluorogenic substrates in HCV assays. For example, WO 97/08194 and WO 9719103 disclose fluorescent substrates for hepatitis C virus NS3 serine protease assays that are derived from amine containing fluorophores. Resonance energy transfer (e.g., DABCYL/EDANS) based fluorescent substrates and 7-amido-4-methylcoumarin (AMC) based fluorogenic substrate are commercially available (BACHEM).
Substrates have also been reported for use in fluorescence polarization assays to detect both viral and non-viral proteases and their inhibitors [jolley et al., 1996, Journal of Biomolecular Screening 1(1):33-38; Levine et al., 1997, Analytical Biochemistry 247:83-88; Schade et al ., 1996, Analytical Biochemistry 243:1-7].
Known chromogenic and fluorogenic substrates, however, lack the sensitivity and/or cleavability necessary for an optimal HCV NS3 protease assay. Since HCV NS3 protease has its distinct substrate specificity [e.g., see Urbani et al, 1997, Journal of Biological Chemistry, 272(14):9204-9209; Zhang et al, 1997, Journal of Virology, 71(8): 6208-6213; Kakiuchi et al, 1997, Journal of Biochemistry, 122: 749-755], the pNA-based chromogenic substrates and AMC-based fluorogenic substrates for HCV NS3 protease, in contrast to similar types of substrates for other proteases, turned out to be very inefficiently cleaved, requiring long reaction time and large quantities of the protease to generate weakly detectable signals. No fluorescence polarization based substrates for HCV NS3 protease have been reported.
In order for a chromogenic, fluorogenic or fluorescence polarization substrate to be practically useful in assays which monitor single end-point or continuous inhibition kinetics and provide rapid characterization of HCV NS3 protease inhibitors, there is a need for the substrates to be highly sensitive and deavable and have high specificity for the HCV NS3 protease.